Forensic Body Fluid Identification Using Epigenetic Markers Hwan Young Lee Department of Forensic Medicine Yonsei University College of Medicine Forensic Sciences o Application of science to solve a legal problem 1
The Hierarchy and DNA Evidence o Case issues are classified as belonging to one of four levels § Sub-source level “From whom has the DNA originated” § Source level “Could the blood present in the evidentiary stain have originated from the defendant?” § Activity level “What actions resulted in the observed blood stain patterns?” § Offence level “Is the defendant guilty of the crime?” o The activity that led to the deposition of the cellular material is more and more assessed Forensic Body Fluid Identification o Knowledge regarding the cell types residing in an evidentiary trace can facilitate inference of activities o Chemical or catalytic tests o Assays for RNA, DNA methylation and microbial markers 2
DNA Methylation across Tissues Blood Blood (Adult) (Infant) Unsupervised clustering of average beta values in normal human tissues. Christensen et al. PLoS Genet (2009) Methylation Profiling of Body Fluids o DNA methylation profiles were produced in 42 body fluid samples including 12 blood, 12 saliva, 12 semen, 3 vaginal fluid and 3 menstrual blood using Human Methylaion450 BeadChip array (GSE59505) Sample Age Sample Age Sample Age Samples Age BL-01 24 SA-01 20 SE-01 20 VF-01 21 BL-02 27 SA-02 24 SE-02 27 VF-02 21 BL-03 28 SA-03 27 SE-03 28 VF-03 23 BL-04 33 SA-04 29 SE-04 31 MB-01 21 BL-05 37 SA-05 32 SE-05 37 MB-02 23 BL-06 38 SA-06 37 SE-06 38 MB-03 27 BL-07 41 SA-07 38 SE-07 41 BL-08 43 SA-08 43 SE-08 43 BL-09 48 SA-09 48 SE-09 48 BL-10 50 SA-10 50 SE-10 57 BL-11 57 SA-11 55 SE-11 59 BL-12 59 SA-12 59 SE-12 59 Lee HY et al. Forensic Sci Int Genet (2015) 3
Human Methylation450 BeadChip Infinium I (Allele-specific) Infinium II (SBE: Single Base Extension) Significant Probes from 450K Array Comparison a Cut-off b No. of CpGs 1 SE vs. BL Abs (delta_mean) ≥ 0.3, fdr. P < 0.05 64,079 SE vs. SA Abs (delta_mean) ≥ 0.3, fdr. P < 0.05 64,305 SE vs. VF Abs (delta_mean) ≥ 0.3, fdr. P < 0.05 54,062 SE vs. MB Abs (delta_mean) ≥ 0.3, fdr. P < 0.05 45,310 BL vs. SA Abs (delta_mean) ≥ 0.3, raw P < 0.05 9,100 BL vs. VF Abs (delta_mean) ≥ 0.3, raw P < 0.05 442 BL vs. MB Abs (delta_mean) ≥ 0.3, raw P < 0.05 556 SA vs. VF Abs (delta_mean) ≥ 0.3, raw P < 0.05 620 SA vs. MB Abs (delta_mean) ≥ 0.3, raw P < 0.05 371 VF vs. MB Abs (delta_mean) ≥ 0.2 , raw P < 0.05 0 SE vs. (BL, SA, VF, MB) Abs (delta_mean) ≥ 0.5 , fdr. P < 0.05 20,542 2 BL vs. (SA, VF, MB) Abs (delta_mean) ≥ 0.3 , raw P < 0.05 4,252 SA vs. (BL, VF, MB) Abs (delta_mean) ≥ 0.3 , raw P < 0.05 2,771 (VF, MB) vs. (BL, SA) Abs (delta_mean) ≥ 0.2 , raw P < 0.05 604 a BL, SA, SE, MB and VF represent blood, saliva, semen, menstrual blood and vaginal fluid, respectively b Abs (delta_mean) represents the discrepancy in the mean values of average β-scores between a certain type of body fluid and the other type of body fluids 4
Body Fluid-Specific CpG Candidates o Body fluid-specific hyper or hypo-methylation with a low standard deviation in the same type of body fluid o Complete methylation or unmethylation in other types of body fluids Selection of Body Fluid-Specific CpGs SE1 SE2 SE3 BL1 BL2 VF1 VF2 SA1 5
Validation of Body Fluid-Specific CpGs o Body fluid-specificity was further investigated using direct sequencing and a SBE reaction of bisulfite-converted DNA Semen Blood Validation of Body Fluid-Specific CpGs o Body fluid-specificity was further investigated using direct sequencing and a SBE reaction of bisulfite-converted DNA Vaginal fluid Saliva Y Y Y Y Y Y Y Y Y Y Y Y 6
Multiplex SBE for Body Fluid Identification Multiplex SBE for Body Fluid Identification o A total of 151 samples were analyzed with the multiplex SBE 7
Body Fluid Specificity of Selected CpGs SE1 SE2 SE3 BL1 BL2 VF1 VF2 SA1 Menstrual Blood and Semen Profiles Menstrual blood (n = 7) Menstrual blood (n = 9) Semen (n = 20) Semen (n = 20) Semen vasectomy (n = 9) 8
Sensitivity of the Developed Multiplex o Successful methylation profiling results could be obtained with approximately 0.5 ng of bisulfite converted DNA Aged Sample Test o Samples exposed to the environment for 75 days produced successful DNA methylation profiling results and correct identification results 9
Artificial Casework Sample Test o Successful methylation profiling results could be obtained with mixed body fluid samples A post-coital vaginal swab A post-coital vaginal sample obtained with clean tissue paper A post-coital penile sample obtained with clean tissue paper A skin swab from a kissing site A swab from clean skin surface Lee HY et al. Forensic Sci Int Genet (2015) Modification of Multiplex SBE o The multiplex SBE was modified by replacing SE3 and BL2 with BL3 (cg08792630), and was registered as a standard operating procedure of NFS Semen Blood Vaginal fluid Menstrual blood Saliva 10
Summary o Multiplex methylation SNaPshot reactions were constructed to differentiate between blood, saliva, semen and vaginal fluid o The multiplex could produce successful methylation profiles in aged or mixed samples o It appears favorable to have multiple markers per body fluid type o DNA methylation-based tools have the potential to not only body fluid identification but also gender and age estimation, discrimination of monozygotic twins, etc. Acknowledgement § Ja Hyun An § Sang-Eun Jung § Eun Young Lee § Ajin Choi § Yu Na Oh § Woo Ick Yang § Kyoung-Jin Shin § Basic Science Research Program § National Forensic Service 11
Thank you for your attention! http://forensic.yonsei.ac.kr hylee192@yuhs.ac 12
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